Trypanosome class II transcription pre-initiation complex

布鲁氏锥虫、克氏锥虫和利什曼原虫分别是人类主要疾病非洲昏睡病、恰加斯病和利什曼病的主因。治疗这些疾病的药物很少而且有毒，寄生虫对这些药物的耐药性正在上升。因此，寻找新的抗寄生虫靶点和开发新的治疗策略变得越来越重要。所有的锥虫依赖于相同的不寻常的基因表达模式，包括蛋白质编码基因的多顺反子转录和核前mrna的反式剪接。在这一过程中至关重要的是寄生虫特异性剪接先导RNA (SL)，其中5 ‘末端部分被切割并融合到每个mRNA的5 ’端。由于SL RNA在这一过程中被消耗，因此寄生虫在其整个生命周期中至关重要地依赖于持续强的SL RNA合成。我们最近在布鲁氏杆菌中鉴定和鉴定了几个对SL RNA基因转录至关重要的蛋白质。这些包括一般转录因子TFIIA， TFIIB， TFIIH和候选TFIIE的极端不同的同源物，这表明寄生虫在SLRNA基因（SLRNA）启动子处形成II类转录预起始复合物（PIC），以招募RNA聚合酶II进行准确的转录起始。这些蛋白和它们的哺乳动物同源蛋白之间的序列差异非常大，这阻碍了它们在完成的Tritryp基因组计划中被识别出来，这表明锥虫的PIC与人类的PIC有很大的差异。为了探索这种可能性，我们将对SLRNA启动子上形成的PIC进行功能表征。这将包括RNA干扰、体外转录实验和dna -蛋白质相互作用分析，以评估鉴定的蛋白质对寄生虫生长的重要性以及在SLRNA转录中的作用。最后，我们将分析寄生虫是否使用PIC形成将RNA聚合酶II募集到其蛋白质编码基因的长串联阵列中。染色质免疫沉淀结合深度测序将鉴定布鲁氏体基因组中非slrna PIC形成位点。总之，这些实验可能揭示了一个基本重要过程中独特和必要的结构和/或生化特征，即RNA聚合酶II向DNA的募集。

The trypanosomatid parasites Trypanosoma brucei, T. cruzi and Leishmania spp. (Tritryp) cause the major human diseases African Sleeping Sickness, Chagas’ disease, and Leishmaniasis, respectively. Drugs for these diseases are few and toxic and parasite resistance to these drugs is on the rise. Thus, it becomes increasingly important to find new anti-parasitic targets and develop new therapeutic strategies. All trypanosomatids depend on the same unusual mode of gene expression involving polycistronic transcription of protein coding genes and trans splicing of nuclear pre-mRNA. Of central importance to this process is the parasite-specific spliced leader (SL) RNA from which the 5’ terminal part is cleaved and fused to the 5’ end of each mRNA. Since SL RNA is consumed in this process, the parasites crucially depend on continuously strong SL RNA synthesis throughout their life cycle. We have recently identified and characterized several proteins in T. brucei which are essential for SL RNA gene transcription. These include extremely divergent homologues of the general transcription factors TFIIA, TFIIB, TFIIH, and a candidate TFIIE suggesting that the parasites form a class II transcription pre-initiation complex (PIC) at the SL RNA gene (SLRNA) promoter to recruit RNA polymerase II for accurate transcription initiation. The extraordinary level of sequence divergence between these proteins and their mammalian orthologues, which prevented their identification in the completed Tritryp genome projects, indicates that the trypanosome PIC deviates substantially from its human counterpart. To explore this possibility, we will functionally characterize the PIC which forms at the SLRNA promoter. This will include RNA interference, in vitro transcription experiments and DNA-protein interaction assays to evaluate the identified proteins’ importance for parasite growth and role in SLRNA transcription. Finally, we will analyze if the parasites use PIC formation to recruit RNA polymerase II to their long tandem arrays of protein coding genes. Chromatin immunoprecipitation in combination with deep sequencing will identify non-SLRNA PIC formation sites in the T. brucei genome. Together, these experiments may uncover unique and essential structural and/or biochemical features in a fundamentally important process, namely the recruitment of RNA polymerase II to DNA.